Combination of selenium-containing compounds with cytostatics

ABSTRACT

The present invention relates to the use of selenium and/or a derivative thereof in combination with one or more cytostatics. 
     It is the object of the present invention to provide a possibility of enhancing the effect of antitumor drugs, and to provide said drugs in a suitable form of administration. 
     Said object is achieved by using selenium and/or at least one selenium compound for enhancing the effect of one or more cytostatics. This combination results in a synergistic, i.e. superadditive, enhancement of the effect. Furthermore, the present invention provides a kit containing selenium and/or at least one selenium compound and one or more cytostatics as a combination preparation for cytostatic therapy. The present invention can be used efficiently against various types of tumor cells, but especially against large-cell and small-cell bronchial carcinomas, adenocarcinomas, pancreatic carcinomas, prostatic carcinomas and hypemephromas.

This application is a 371 of PCT/EP99/03771 filed May 31, 1999.

The present invention relates to the use of selenium and/or a derivativethereof in combination with a cytostatic or a mixture of cytostatics.

The chemical element selenium is a trace element which is essential forhumans and animals and influences above all oxidative processes as wellas thyroxine metabolism. In humans it could be detected that the enzymeglutathione peroxidase and the selenoprotein P found in plasma containselenium in the form of the amino acid selenocysteine. Theselenium-containing glutathione peroxidase forms part of theantioxidative protective system of the mammalian cell. In the presenceof sufficient amounts of substrate, i.e. reduced glutathione,glutathione peroxidase converts a multitude of different hydroperoxidesinto corresponding alcohols. It could be demonstrated that the integrityof cellular and subcellular membranes decisively depends on theintactness of the glutathione peroxidase system. Selenium as part ofglutathione peroxidase can reduce the lipid peroxidation rate and theresulting membrane damage.

In animals the type-I iodothyronine-5′-deiodase was recentlycharacterized as a selenium-containing enzyme. In the thyroid, liver andlung of humans, iodothyronine deiodase also converts thyroxine (T₄) intotriiodothyronine (T₃), the active thyroid hormone. In the case ofselenium deficiencies, e.g. phenylketonuria and cystic fibrosis,increased T₄ values could be detected at a simultaneously reduced T₃level. By the administration of sodium selenite (Na₂SeO₃) the thyroidmetabolism is normalized again.

As a further selenium-dependent enzyme, a human thioredoxin reductasefrom lung cells was recently described to contain selenium as a cofactor(Tamura and Stadtman, 1996, Biochemistry, Proc. Natl. Acad. Sci., 93:1006-101 1). The enzyme could so far be isolated from T cells, lungtissue and placenta (Gladyshev et al., 1996, Biochemistry, Proc. Natl.Acad. Sci., 93: 6146-6151). The selenium-dependent enzyme thioredoxinreductase reduces thioredoxin. Thioredoxin is overexpressed in a numberof tumors, and some experimental studies have shown that thioredoxincontributes to the growth and malign transformation of some human cancercells. The enzyme thioredoxin reductase therefore plays a role in theregulation of the growth of normal and cancer cells.

Proof of the pathophysiological relevance of the selenium-dependentreactions has been furnished by observation of selenium deficiencysymptoms in humans and in animals. Deficiency of this trace elementintensifies oxidatively or chemically induced liver damage and thetoxicity of heavy metals such as mercury and cadmium.

In humans the Keshan disease, an endemically occurring cariomyopathy,and the so-called Kaschin-Beck disease, also an endemically occurringosteoathropathy with strong deformations of the joints, are described asselenium deficiency symptoms. Clinically manifested selenium deficiencywas also observed as a consequence of long-term parenteral feeding andof balanced diets. Cardiomyopathies and myopathies of the skeletalmuscles as well as a shift in the T₃/T₄ ratio were above all observed.

Epidemiological studies hint at an inverse correlation betweenblood-selenium level and the incidence of cardiovascular diseases(cardiomyopathies, arteriosclerosis, myocardial infraction) and tumordiseases, in particular of the digestive system, breast and liver.Reduced selenium levels in plasma may be present in patients with renalinsufficiency and in the case of gastrointestinal diseases. Seleniumdeficiency can be detected through a reduced selenium level in wholeblood or plasma and a reduced glutathione peroxidase activity in wholeblood, plasma or thrombocytes.

Selenium substitution in the case of deficiency symptoms activatesreactions of the immune defense, in particular unspecific, cell-boundand humoral reactions. The selenium-containing glutathione peroxidaseinfluences leukotriene, thromboxane and prostacyclin metabolism. Theimmunomodulatory effects of selenium-containing compounds are listed inthe following:

Stimulation of lymphocyte proliferation

Activation of cytotoxic T cells and NK cells

Increase in interleukin-2 receptor expression

Selective reduction of the number of T suppressor cells

Increase in interferon-γ synthesis

general decrease in infection frequency

Selenium in the form of selenite (SeO₃ ²⁻) is not directly incorporatedinto proteins. In blood, selenite is first mainly taken up byerythrocytes and enzymatically reduced to selenium hydrogen. Seleniumhydrogen serves as a central selenium pool for excretion and for thetargeted incorporation into selenoproteins. In this reduced formselenium is bound to plasma proteins which migrate into the liver andother organs. The plasmatic secondary transportation starting from theliver into the glutathione peroxidase-synthetized target tissue probablytakes place in the form of a selenocysteine-containing P selenoprotein.The further metabolic course of selenoprotein biosynthesis has so faronly been known from prokaryotic model organisms. In these organismsselenocysteine is specifically incorporated-into the peptide chain ofthe glutathione peroxidase in the course of the translation.

Excessive selenium hydrogen in humans is metabolized throughmethylselenol and dimethylselenide to trimethylselenonium ion, the mainexcretion product. After oral application selenite is predominantlyabsorbed from the small intestine. The intestinal absorption of sodiumselenite is not regulated homeostatically. Depending on theconcentration and on additives, it is between 44% and 89%, sometimesover 90%. The amino acid cysteine promotes the sodium seleniteabsorption.

Organic selenium compounds must first be converted into seleniumhydrogen before they are available for the synthesis of selenoproteins.Instead of methionine, selenomethionine, which is mainly contained infood, can also be unspecifically incorporated statistically in the caseof protein biosynthesis into proteins that do not contain selenium.

The total amount of selenium in the human body is between 4 mg and 20 mgin a well-balanced selenium metabolism. Selenium is excreted in humansvia urine, faeces and lung, depending on the dose applied. Selenium isprimarily excreted renally in the form of the above-mentionedtrimethylselenonium ions.

In humans acute selenium intoxications have hardly been described up tonow. Garlic-like breadth, tiredness, queasiness, diarrhea and abdominalpain are regarded as signs of an acute overdosage. In humans, a safemaximum daily intake of selenium of 820 μg was inferred fromobservations regarding the chronic toxicity of selenium, while a dosageof up to 500 μg per day is also considered to be harmless in sensitivepersons. As clinical signs of endemically occurring selenosis, alopecia,brittleness of the finger nails, skin alterations and disorders in thenerve system were observed in a study carried out in China after a dailysupply of 3200-6700 μg selenium. In various species a decreasedreproductive capacity because of a reduced motility of spermatozoons wasdescribed as a symptom of selenosis.

In a dose/escalation study, between 10 and 50 mg selenium were infusedin the form of sodium-selenite pentahydrate in tumor patients. Within 30minutes the selenium level in plasma rose from 200 μg/l to 1200 μg/lafter administration of 10 mg selenium as sodium selenite. After 8 and16 hours the plasma selenium decreased to 770 μg/I and 430 μg/I,respectively. After 24 hours the selenium level in plasma had againreached its initial value. Gastrointestinal toxicity was observedstarting from about 20 mg selenium as sodium selenite and was reversibleafter the administration of the preparation had been stopped (Röhrer H.,1989, Erfahrungsheilkunde 38: 10a, 761).

As counter-measures in the case of intoxication, gastric lavage, forceddiuresis, or highly dosed vitamin C administrations are possible. In thecase of an extreme overdosage (1000 to 10000 times), the attempt can bemade to eliminate selenite by dialysis.

In humans, the trace element selenium is predominantly taken in byconsumption of yolk, fish and meat, in particular chicken and pork, aswell as innards. The minimum selenium supply required for humans dependson the chemical form of the consumed element and on the composition ofthe diet in which it is present. In China, experiments revealed anamount of 15-20 μg selenium a day to be sufficient as protection againstendemic selenium deficiency diseases. The National Research Council(NRC) of the USA recommends a daily supply of 70 μg selenium for malesand 55 μg selenium for females. In former times (up to 1989) the NCRregarded daily amounts of 50-200 μg selenium as adequate and harmless.The German Society for Alimentation recommends 20-100 μg selenium perday.

The daily average selenium supply, 2/3 covered by the supply of animalprotein, is 38 μg for women and 47 μg for men in the old federal statesof Germany. By contrast, in the territory of the new federal states ofGermany, values of 20-25 μg selenium were determined. These figuresdemonstrate that the nutritive selenium supply in Germany is not alwayscovered. The risk of an insufficient supply with selenium existsespecially in situations of increased demands (e.g. pregnancy andlactation period), in persons exposed to heavy metals and oxidants, inpatients with gastrointestinal complications (e.g. chronicallyinflammatory bowel diseases) and in parenterally fed persons or personsobserving special diets (e.g. in the case of phenylketonuria).

Epidemiological studies have shown that a low selenium intake andcorrespondingly low selenium levels in plasma are connected with anincreased incidence of a variety of cancers in humans (Glattre et al.,1989, Int. J. Epedemiol., 18:45-49; Knekt et al., 1990; J. Natl. CancerInst., 82:864-868; Burney et al., 1997, J. Clin. Nutr., 49:895-900).Selenium has also been shown to markedly inhibit the growth of differenttumor cells in vitro in high dose levels (20-200 μM), including humanmammary, ovarian and colon tumor cells. (Yan et al., 1991; Biol. TraceElem. Res., 30:145-162; Chen et al., 1995, FASEB J., 9(3): A159; Nano etal., 1989, Biol. Trace Elem. Res. 20: 31-43; Stewart et al., 1997,Cancer Lett., 117:35-40). By contrast, several scientists reported onthe growth stimulating effect of small amounts of sodium selenite(0.001-1 μM) on various tumor cells incubated under serum-free cultureconditions (Nano et al., 1989, Biol. Trace Elem. Res., 20:31-43;Goiczewski and Frenkel, 1989, Biol. Trace Elem. Res. 20:115-126). It hasalso been observed that organic selenium compounds have a preventiveeffect on the tumor development of mammary carcinomas in mice and rats(El-Bayoumy et al., 1995, J. Cell. Biochemistry, Annex 22: 29-100). Themechanism by which selenium influences tumor proliferation or regressionis mainly unknown. However, it seems that induction of DNA strand breaksand apoptosis due to selenium and/or selenium metabolites likeselenodiglutathione and hydrogen selenite as well as the formation ofselenoproteins such as glutathione peroxidase and thioredoxin reductaseplay an important role (Thompson et al., 1994, Carcinogenesis15:183-186; Wu et al., 1995, Carcinogenesis 16: 1579-1584; Lu et al.,1994, Biochem. Pharmacol., 47:1531-1535; Milner, 1985, Fed. Proc., 44:2568-2572; Schrauzer, 1992, Biol. Trace Elem. Res., 33:51-62; Gallegoset al., 1997, Cancer Res., 75:4965-4979). For example, enhancingthioredoxin reductase activity by selenium could reduce cellularthioredoxin concentration and therefore play a role in the growthregulation of cancer cells (Gallegos et al., 1997, Cancer Res., 75:4965-4979).

In combination experiments it has been observed that the administrationof small amounts of selenium or selenium-containing compounds togetherwith cytostatics does not decrease the antitumor effect, but canconsiderably reduce the side effects caused by cytostatics, for instancenephrotoxicity or cardiotoxicity.

While quite efficient therapeutic methods could already be developed forsome types of cancer (the mortality rate following colon cancer diseasecould e.g. be reduced by about 17% between 1992 to 1993), there has sofar been no or only a very inadequate therapy for the great majority oftypes of cancer.

Apart from the operative removal of the tumor and radiation therapy,chemotherapy is considered the so far most efficient therapeutic method.Chemotherapeutic drugs can substantially be divided into the followingfour groups: antimetabolites, topoisomerase inhibitors, alkylatingagents and plant alkaloids, the three first-mentioned groups preventinga correct replication of the genetic substance, and the last-mentionedgroup having a mitosis-inhibiting effect. In the treatment of above allsolid tumors the effect of cytostatics is most of the time notsufficient for curatively treating tumors.

It is therefore the object of the present invention to provide apossibility of enhancing the effect of antitumor drugs and to providesaid drugs in a suitable form of administration.

This object is achieved by using selenium and/or at least one seleniumcompound for enhancing the effect of a cytostatic or a mixture ofcytostatics.

Furthermore, this object is achieved by providing a kit comprisingselenium and/or at least one selenium compound and a cytostatic or amixture of cytostatics as a combination preparation for simultaneous,separate or sequential application in cytostatic therapy.

The present invention relates to the use of selenium and/or at least oneselenium compound for enhancing the effect of a cytostatic or a mixtureof cytostatics. The following examples will demonstrate that in vitro asimultaneous treatment with the above-mentioned components surprisinglyyields a distinct synergistic, i.e. superadditive, antitumor effect.

Organic and inorganic selenium compounds are used for combination withcytostatics. In a preferred embodiment use is made of an organicselenium compound. The use of organic selenium compounds is to reducetoxicity in comparison with inorganic selenium compounds withsimultaneous or improved antitumor efficiency. Particularly preferredare the selenium amino acids selenomethionine and selenocysteine as wellas the compound phenylenebis(methylene)selenocyanate as well asderivatives thereof (El-Bayonmi et al., 1995, J. Cell. Biochemistry,Annex 22: 92-100). The last-mentioned compound inhibits thymidine kinasein human mammary carcinoma cell lines. Furthermore, it has been reportedthat said compound can trigger the inhibition of cell growth and theinduction of cell death by apoptosis.

Furthermore, a selenium oxide is preferred as the selenium compound forenhancing the effect of a cytostatic or a mixture of cytostatics. In aparticularly preferred embodiment, the selenium compound is a salt ofSeO₂, e.g. the salt Na₂SeO₃.

The cytostatic that is used together with selenium or a seleniumcompound may be a mitosis-inhibiting cytostatic. Examples of said groupare inter alia substances, such as vinblastine and vinorelbine.

The cytostatic used together with selenium or a selenium compound mayalso be a cytostatic inhibiting nucleic acid synthesis, for examplemethotrexate and fluorouracil, which belong to the group ofantimetabolites, or the topisomerase inhibitor topotecan, mRNA synthesisinhibitors such as doxorubicin, or alkylating agents such ascyclophosphamide and chlorambucil. The following table gives examples ofdifferent cytostatics, in the order of their modes of action, which aresuited for administration together with selenium compounds. Combinationsof several different cytostatics can also be used together with seleniumcompounds.

Effect Groups of cytostatics and on Mode of action examples ofsubstances Dosage ranges DNS Enzyme inhibition Antimetabolites bio--dihydrofolate re- Methotrexate 20-40 mg/m²/d i.v. syn- ductase HD: 12g/m² thesis -thymidylate 5-FU 500-600 mg/m² i.v. or 2-2,6 synthase g/m²i.v. (24-h-inf) DNS polymerase ZD1694 (Tomudex) 3 mg/m²/d i.v.Capecitabine about 500 mg/m²/d p.o. Gemcitabine 1000-1250 mg/m² i.v.Cytosine arabinoside 200 mg/m²/d i.v. HD: 3 g/m² -ribonucleotide re-Hydroxy urea 800-1600 mg/m²/d p.o. ductase 6-mercaptopurine 100 mg/m²/dp.o. DNS Induction of strand Alkylating agents breaks -intermediatestrand Mustargen 6 mg/m² cross-linkage -intercalation Estramustinephosphate 480-550 mg/m²/d p.o. 150- 200 mg/m²/d i.v. Melphalan 8-10mg/m²/d p.o. 15 mg/m²/d i.v. Chlorambucil 3-6 mg/m²/d p.o. Prednimustine40 mg/m²/d p.o. or 60-100 mg/m²/d p.o. Cyclophosphamide 750-1200 mg/m²/di.v. or 50- 100 mg/m²/d p.o. HD: 2,4 g/m² i.v Ifosfamide 1500-2000mg/m²/d i.v. Trofosfamide 150-200 mg/m²/d p.o. initially then 25-100mg/m² Busulfan 2-6 mg/m²/d p.o. Treosulfan 5-8 g/m²/d i.v. or 1500mg/m²/d p.o. 12-16 mg/m² i.v. Thiotepa 100 mg/m²/d i.v. Carmustine 100mg/m² p.o. Lomustine 90-100 mg/m² i.v. Nimustine 100-200 mg/m²/d i.v.Dacarbazine 100 mg/m²/d p.o. Procarbazine 1.5 mg/m²/d i.v.-Topoisomerase Topotecan 100-350 mg/m² i.v. toxins Irinotecan Platinumcomplexes 20 mg/m²/d i.v. or 80-120 Cisplatin mg/m²/d i.v. 300-400 mg/m²i.v. Carboplatin 80-135 mg/m² i.v. Oxaliplatin Antibiotics (see below)Epipodophyllotoxins 100-200 mg/m²/d i.v. Etoposide 20-30 mg/m²/d i.v.Teniposide RNA Blockage of mRNA Antibiotics synthesis by Aclarubicin25-100 mg/m² i.v. intercalation Bleomycin 10-15 mg/m² i.v. Actinomycin D(Dactino- 0,6 mg/m²/d i.v. mycin) Incorporation in RNA Daunorubicin45-60 mg/m²/d i.v. Doxorubicin 45-60 mg/m² i.v. Epirubicin 60-80 mg/m²i.v. Idarubicin 10-12 mg/m² i.v. or 35-50 mg/m² p.o. Mitoxantrone 10-12mg/m² i.v. Mitomycin C 10-20 mg/m² i.v. Antimetabolites (see above) Pro-Modifications of Hormones & Antagonists tein receptor bindings Vitamin-Aacid deriv. Tretinoin 45 mg/m² Inhibition of Vinca alkaloids tubulinpoly- merization Vincristine 1,5-2,0 mg i.v. Vindesine 2-3 mg/m² i.v.Vinblastine 4-8 mg/m² i.v. Vinorelbine 25-30 mg/m² Taxanes Taxol(Paclitaxel) 175 mg/m² i.v. Taxotere (Docetaxel) 100 mg/m² i.v. Proteincross-linkage Alkylating agents (see Phosphorylation above) Proteinkinase-C inhibi- tors Miltefosine max. 5 ml/d, local application HD =high-dose therapy

A preferred cytostatic inhibiting nucleic acid synthesis is gemcitabine.A further, also preferred, cytostatic that inhibits nucleic acidsynthesis is the compound mitomycin C. The structural formulae of saidtwo compounds are shown in the following:

Mitomycin C belongs to the group of alkylating agents. Upon reduction ofthe quinone unit, methanol is released, which facilitates opening of theaziridine ring to form an alkylating metabolite. A further alkylatingmolecule is formed by chemical or enzymatic separation of the carbamateside chain. Moreover, the reduction of the quinone unit is connectedwith the formation of reactive oxygen molecules, which also havealkylating potency. The antitumor effect of mitomycin is mainly due tothe alkylation of DNA.

Gemcitabine is a pyrimidine antimetabolite. After cellular uptake, it ismetabolized to 2′,2′-difluoro-deoxycytidinetnphosphate. Incorporation ofgemcitabine into DNA terminates DNA strand synthesis, so that celldivision is no longer possible.

Gemcitabine and mitomycin C have different modes of action, but bothcompounds interact directly with cellular DNA, leading to errors ordiscontinuance of DNA replication.

To enhance the effect of a cytostatic or the combination of severalcytostatics, selenium and/or at least one selenium compound is used in aconcentration of 0.1 mg/kg body weight to 1.25 mg/kg body weight, andthe cytostatic is used in a concentration of 2 mg/M² body surface to 240g/m² body surface. The preferred concentration of selenium or a seleniumcompound is in a range of 0.1 mg/kg body weight to 0.3 mg/kg bodyweight, and that of the cytostatic is in a range of 20 mg/M² bodysurface to 1000 mg/m² body surface.

The use of said combination in cytostatic therapy is particularlypreferred.

Furthermore, the invention provides a kit which comprises seleniumand/or at least one selenium compound and a cytostatic or a mixture ofcytostatics as combination preparation for simultaneous, separate orsequential application in cytostatic therapy. It is preferred that theselenium compound contained in the kit is an organic selenium compound.Particularly preferred organic selenium compounds are the selenium aminoacids selenomethionine and selenocysteine as well as the compoundphenylene-bis(methylene)selenium cyanate. Moreover, a selenium oxide ispreferred in a further embodiment. Particularly preferred is a salt ofSeO₂, e.g. Na₂SeO₃.

Furthermore, the above-mentioned kit may contain a cytostatic which is amitosis-inhibiting cytostatic, e.g. selected from the above-mentionedcompounds. Furthermore, the cytostatic may also be a cytostaticinhibiting nucleic acid synthesis. The compounds gemcitabine andmitomycin C are here particularly preferred cytostatics inhibitingnucleic acid synthesis.

The kit according to the invention contains selenium and/or at least oneselenium compound in a concentration of 0.1 mg/kg body weight to 1.25mg/kg body weight and a cytostatic as described above in a concentrationof 2 mg/M² body surface to 240 mg/M² body surface. A concentration rangeof 0.1 mg/kg body weight to 0.3 mg/kg body weight is here preferred forselenium or a selenium compound, and a concentration range of 20 mg/kgbody weight to 1000 mg/M² body weight of a cytostatic as characterizedabove.

The combinations of a selenium compound and a cytostatic or severalcytostatics can be administered in solid or liquid form. The applicationmay be oral, rectal, nasal, topical (including buccal and sublingual),vaginal or parenteral (including intramuscular, subcutaneous andintravenous), or by inhalation. They may be administered together withconventional adjuvants, carriers and/or diluents.

The solid forms of application comprise tablets, capsules, powders,pills, pastilles, suppositories and granular forms of administration.They may also include additives, such as flavors, dyes, diluents,softeners, binders, preservatives, blasting agents and/or enclosingmaterials.

Liquid forms of administration include solutions, suspensions andemulsions. These may also be offered together with the above-mentionedadditives.

The following figures and examples will explain the present invention:

FIG. 1: Effect of sodium selenite (3 μM and 30 μM) on the colonyformation of pancreatic tumor xenografts (PAXF 546 and 736) in theclonogenic assay in vitro in different experiments. T/C=number of tumorcolonies in the selenium-treated samples divided by number of coloniesin the untreated samples in %.

FIG. 2: Effect of selenium and mitomycin C given alone or simultaneouslyon the in vitro growth of the pancreatic tumor xenograft PAXF 546(clonogenic assay, continuous drug exposure). A-C: Dose/response curvesof mitomycin C und mytomycin C in combination mit 3 μM selenium (A) or30 μM selenium (B, C). The effect of selenium alone is indicated by thedashed lines.

FIG. 3: Comparison of the experimentally observed and expected T/Cvalues for seleniumlmitomycin C combinations. Expected T/C values for anadditive drug effect are shown as open bars, experimentally observed T/Cvalues as solid bars;

indicates a significant difference (p<0,01) between T/C_(expected) andT/C_(observed) and therefore drug synergism. A-C: Combination ofmitomycin C with 3 μM selenium (A) or 30 μM selenium (B, C).

FIG. 4: Effect of selenium gemcitabine given alone or in combination onthe in vitro growth of the pancreatic tumor xenograft PAXF 546(clonogenic assay, continuous drug exposure). A-C: Dose/response curveof gemcitabine alone and gemcitabine in combination with 3 μM selenium(A) or 30 μM selenium (B, C). The effect of selenium alone is indicatedby the dashed lines.

FIG. 5: Comparison of the experimentally observed and expected T/Cvalues of selenium/gemcitabine combinations. Expected T/C values for theadditive drug effect are shown as open bars, experimentally observed T/Cvalues as solid bars;

indicates a significant difference (p<0,01) between T/C_(expected) andT/C_(observed) and therefore drug synergism. A-C: Combination ofgemcitabine with 3 μM selenium (A) or 30 μM selenium (B, C).

EXAMPLES 1. Material and Methods

Sodium selenite (Selenase®) was provided by G. N. Pharm GmbH, Fellbach,Germany. The 0.9% NaCl solution contained 50 μg/ml (0,63 mM) of seleniumas Na₂SeO₃×5H₂O. Gemcitabine and mitomycin C were purchased from thepharmacy and used as clinical formulations.

1.1 Colony Forming Assay

Preparation of a Single Cell Suspension From Human Tumor Xenografts

Solid human tumor xenografts growing subcutaneously in serial passagesin thymus-aplastic nude mice (NMRI nu/nu strain, obtained from our ownbreeding facility) were removed under sterile conditions, the cells weremechanically disaggregated and subsequently incubated with an enzymecocktail consisting of collagenase (1.2-1.8 U/ml, Worthington), DNAse(375 U/ml, Boehringer Mannheim) und hyaluronidase (29 U/ml, BoehringerMannheim) in RPMI 1640 at 37° C. for 30 minutes. The cell mixture waspassed through sieves of 200 μm and 50 μm mesh size and washedthereafter twice with PBS. The percentage of viable cells was determinedin a Neubauer counting chamber using trypan blue exclusion.

1.2 Culture Methods

The clonogenic assay was performed according to a modified two-layersoft agar assay described by Hamburger and Salmon (Hamburger and Salmon,Science, 197: 461463, 1977). The bottom layer consisted of 0.2 mlIscoves' Modified Dulbecco's Medium with 20% fetal calf serum and 0.75%agar. 8×10³ to 1,6×10⁴ cells were added to 0.2 ml of the same culturemedium together with 0.4% agar and placed in already coated 24-multiwelldishes. Test substances were applied by continuous exposure (drugoverlay) in 0.2 ml medium. Every dish included six control wellscontaining the vehicle- and drug-treated groups in triplicate at sixconcentrations.

Cultures were incubated at 37° and 7% CO₂ in a humidified atmosphere for5 to 15 days (depending on the doubling time of the tumor stem cells)and monitored for colony growth using an inverted microscope. Withinthis period, in vitro tumor growth led to the formation of colonies witha diameter of ≧50 μm. At the time of maximum colony formation countswere performed with an automatic image analysis system (OMNICOM Fas IV,Biosys GmbH). 24 hours prior to this assay, vital colonies were stainedwith a sterile aqueous solution of2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (1mg/ml, 100 μI/well). Drug effects were expressed in terms of thepercentage of surviving cells, obtained by comparison of the mean numberof colonies in the treated plates with the mean colony count of theuntreated controls (test-versus-control-group value, T/C=colonycount_(Treated Group)*100/colony count_(Control Group)).

An assay was considered as significant if the following quality controlcriteria were fulfilled:

Mean number of colonies in the control group dishes for 24-multiwells≧20 colonies with a colony diameter of ≧50 μm.

Colony survival number in wells treated with the positive referencecompound 5-fluorouracil (at the toxic dose of 1000 μg/ml) <30% of thecontrols.

Coefficient of variation in the control group ≦50%. IC₅₀ and IC₇₀ valueswere determined by plotting compound concentration versus cell survivalrate. Mean IC₅₀ and IC₇₀ values were calculated according to theformula:${{mean}\quad {IC}_{50}\quad {and}{\quad \quad}{IC}_{70}} = \frac{\sum\limits_{x = 1}^{n}\quad {\log \quad \left( {IC}_{50,70} \right)_{x}}}{n}$

with x=specific tumor xenograft and n=total number of tumor xenograftsstudied. If an IC₅₀ or IC₇₀ value could not be determined within theexamined dose range, the lowest or highest concentration studied wasused for the calculation.

1.3 Combination Studies

The inhibition of colony formation by the chemotherapeutic agentsgemcitabine and mitomycin C alone as well as in combination withselenium was studied in pancreatic human tumor xenografts PAXF 546 and736 applying the clonogenic assay as described above. Each experimentincluded the chemotherapeutic agents in 6 concentrations, selenium in 2concentrations (3 and 30 μM), and the combination of all doses withselenium at 3 and 30 μM. All compounds and combinations were studied intriplicates. Concentrations of the chemotherapeutic agents were chosenin such a way that T/C values of 0 to 100% resulted therefrom. Eachexperiment was performed twice. The effect of a combination of compoundswas determined by comparing the experimentally observed T/C value of aselenium/cytostatic combination (T/C_(observed)(A+B)) with the expectedT/C value for this combination (T/C_(expected)(A+B)), calculated bymultiplying the number of surviving colonies obtained after treatment ofthe cells with the respective individual substances (T/C (A) und T/C(B)) using the following equation (multiplication method, for exampledescribed by Berenbaum, 1989, Pharmacol. Rev., 41:93-141):

T/C _(expectd)(A+B)=T/C(A)×T/C(B)/100

For a zero-interactive drug combination (additivity of drug effects)T/C_(expected)(A+B)×T/C_(observed)(A+B). A drug combination issynergistic if T/C_(observed)(A+B) is statisticallysignificant>T/C_(expected)(A+B). Statistical significance was determinedby the T-test.

2. Results

2.1. In Vitro Antitumor Activity of Sodium Selenite in Human TumorXenografts

The cytotoxic activity of sodium selenite was studied on the followinghuman tumor xenografts using the clonogenic assay:

TABLE 1 Human tumor xenografts examined Tumor Original Histology LungLXFL 529 non small cell bronchial carcinoma LXFS 650 small cellbronchial carcinoma Breast MAXF 401NL adenocarcinoma, estrogen receptornegative, progesterone receptor negative MAXF adenocarcinoma, estrogenreceptor negative, MCF7X progesterone receptor negative Ovary OVXF 899adenocarcinoma OVXF 1353 adenoid carcinoma Pancreas PAXF 546adenosquamous carcinoma PAXF 736 adenocarcinoma Prostate PC3MXadenocarcinoma DU145X carcinoma Kidney RXF 393 hypernephroma RXF 944LXhypernephroma

Sodium selenite concentrations between 0.001 and 100 μM were applied.T/C values obtained in the various tumor xenografts are shown in Table2. IC₅₀ and IC₇₀ values as well as an IC₇₀ bar diagram, which shows thesensitivity of the various tumors to selenium treatment, are given inTable 3.

Inhibition of the colon formation depends on the selenium dose. Withamounts up to 1 μM no distinct cytotoxic effect could be observed. At 10μM selenium T/C values between 30% were obtained in 2 of 12 xenografts(LXFL 529, PRXF DU145X). At very high selenium concentrations of 100 μM,T/C values below 20% were obtained in all xenografts, which points at anunspecific cytotoxic effect.

TABLE 2 In vitro effect of sodium selenite in human tumor xenograftsTUMOR/ PASSAGE EXP. Test/control (%) at drug concentration [μM] No. No..001 .01 .1 1. 10. 100. LXFL 529/18 X182AM 90− 78− 72− 61−  9+++  0+++LXFS 650/9 X199AM 100−  100−  98− 84− 109− 16++ MAXF 401/16 X186AM 67s− 81s−  53− 74−  66−  8+++ MCF7X/28 X218AM 93− 85− 90− 78−  97−  0+++ OVXF899/33 X185AM 80− 79− 66− 70−  73−  1+++ 1353/17 X217AM 96− 91− 100− 90−100−  0+++ PAXF 546/2 *(2) 82− 112−  100− 104−  88−  2+++ 736/17 X184AM87− 77− 94− 88−  90−  4+++ PRXF PC3M/3 X189/AM 91− 91− 89− 85−  38+ 1+++ DU145X/15 X229AM 97− 96− 92− 91−  5+++  0+++ RXF 393/9 X194AM 62−45− 31+ 31+  32+ 10+++ 423/16 X183AM 99− 111−  120− 92− 110−  0+++active(++, +++)/total 0/12 0/12 0/12 0/12 2/12 12/12 xenografts only 0%0% 0% 0% 17% 100% Table legend: LXF lungs, A adeno, L large cell, Ssmall cell cancer xenograft; MAXF breast cancer xenograft; OVXF ovarycancer xenograft, PAXF pancreas, PRXF prostate cancer xenograft; RXFkidney cancer xenograft − (T/C ≧ 50%); + (30% ≦ T/C < 50%); ++ (10% <T/C < 30%); +++ (T/C ≦ 10%), s result of a dish

In the IC₇₀ bar diagram (IC₇₀ plot, Table 3) variations of individualIC₇₀ values from the mean value are expressed as bars in the logarithmicrepresentation. Bars to the left demonstrate IC₇₀ values lower than themean value, bars to the right demonstrate higher values. The IC₇₀ plotrepresents therefore a characteristic antiproliferative profile of thecompound.

The mean IC₅₀ of sodium selenite was 15.5 μM, the mean IC₇₀ value 27 μM.This corresponds to a selenium concentration of 1,2 μg/ml (mean IC₅₀)and 2.1 μg/ml (mean IC₇₀). Compared with the efficiency of standardchemotherapeutic agents in this assay, the values are within the scopeof the IC₅₀ and IC₇₀ values of the alkylating agents ifosfamide andcyclophosphamide. Most of the other standard alkylating agents have meanIC₇₀ values of <0,1 μg/ml.

The most sensitive tumor xenografts, represented by IC₇₀ values and barsto the left in Table 3, were the large lung cell cancer xenograft LXFL529, the kidney cancer xenograft RXF 393 and the prostate xenograft PC3Mand DU145X.

TABLE 3 In vitro effect of sodium selenite in human tumor xenografts

2.2 In Vitro Combination Studies

To examine whether selenium given as sodium selenite can potentiate theantiproliferative effect of standard chemotherapeutic agents, twopancreatic human tumor xenografts (PAXF 546 und PAXF 736) were exposedto gemcitabine, mitomycin C or sodium selenite alone or to sodiumselenite combined with one of the two chemotherapeutic agents.

Fixed concentrations of sodium selenite (3 or 30 μM) were administeredto the cells together with 6 different concentrations of thechemotherapeutic agents. According to the data shown in Table 2, asodium selenite concentration of 3 μM was expected to influence colonyformation of the two pancreatic tumors only marginally. This wasconfirmed by the results when sodium selenite was used as a single agentat this concentration in the combination studies (FIG. 1). The highersodium selenite concentrations of 30 μM should have a stronger influenceon tumor colony formation, since the tumor colony formation rate of bothpancreactic tumors rapidly decreases when the sodium seleniteconcentration increases from 10 to 100 μM (Table 2). This could beproven in the further experiments in FIG. 1. The variation of T/C valuesat 30 μM in the two experiments (FIG. 1) is caused by the very steepdose/response values of sodium selenite at concentrations around 30 μM.Therefore, combination experiments with PAXF 546 at this seleniumconcentration were evaluated separately. In the case of PAXF 736 onlyexperiment X265 could be used for evaluation since in experiment X290 30μM of selenium were already strongly cytotoxic with a T/C value of 5%.

The following Tables 4a to 4d show the results of incubation of twopancreatic xenografts with different concentrations of selenium and thecytostatics mitomycin C and gemcitabine. To determine whether a drugcombination has an additive effect of the individual drugs, theexperimentally determined T/C values for a specific combination werecompared with the values expected for an additive effect (see Materialsand Methods). When the pancreatic tumor xenograft PAXF 736 was treatedsimultaneously with sodium selenite and mitomycin C or gemcitabine, nosynergistic effect could be detected, neither at a concentration of 3 μMnor at a concentration of 30 μM sodium selenite. Also no synergism wasobserved between 3 μM sodium selenite and all of the testedconcentrations of chemotherapeutic agents in the xenograft PAXF 546 orwhen low concentrations of the chemotherapeutic agents were combinedwith 30 μM of sodium selenite. In contrast, when higher doses ofchemotherapeutic drugs at which cytotoxic effects could be observed werecombined with 30 μM sodium selenite, synergism was observed incombination with mitomycin C and gemcitabine (Tables 4a, 4b).

TABLE 4a Inhibition of colony growth of the pancreatic cancer xenograftPAXF 546 by Na₂SeO₃ or mitomycin C alone or in combination in vitroSodium selenite Conc. T/C in T/C in % at a mitomycin C concentration(μM) of Treatment in μM % 1 × 10⁶ 1 × 10⁻⁵ 1 × 10⁻⁴ 1 × 10⁻³ 1 × 10⁻² 1× 10⁻¹ 1.0 Synergism Mitomycin C (X261, X295) 87 ± 11 84 ± 14 70 ± 10 70± 13 80± 12 57 ± 8 5 ± 2 Mitomycin C + expect. obs.* 3.0 91 ± 8 84 ± 1584 ± 13 72 ± 12 66 ± 10 60 ± 8 60 ± 10 4 ± 2 no 3 μM Se (X261, X295)expect.** 76 ± 12 76 ± 15 64 ± 11 64 ± 14 73 ± 13 52 ± 9 5 ± 3 MitomycinC + expect. obs. 30.0  80 ± 13 69 ± 10 58 ± 8 49 ± 7 50 ± 8 35 ± 6*** 3± 2 partial 30 μM Se (X261) expected 70 ± 14 67 ± 16 56 ± 14 56 ± 15 64± 14 46 ± 10 expect. obs. 30.0 49 ± 6 38 ± 8 37 ± 6 31 ± 5 30 ± 6 26 ± 514 ± 3 partial (X295) expected 43 ± 12 41 ± 15 34 ± 11 34 ± 14 39 ± 1228 ± 9 *experimentally observed T/C values for the combination ofmitomycin C and selenium **expected T/C values for the combination,calculated by the method: T/C_(expected) = T/C_(se) × T/C_(drug)/100;T/C:T/C value in % with a given treatment at a given concentration***synergistic drug concentrations are marked in italics.

TABLE 4b Inhibition of colony growth of the pancreatic cancer xenograftPAXF 546 by Na₂SeO₃ or gemcitabine alone or in combination in vitroSodium selenite Conc. T/C in T/C in % at a gemcitabine concentration(μM) of Treatment in μM % 1 × 10⁶ 1 × 10⁻⁵ 1 × 10⁻⁴ 1 × 10⁻³ 1 × 10⁻² 1× 10⁻¹ 1.0 Synergism Gemcitabine (X261, X295) 85 ± 15 73 ± 20 80 ± 12 83± 12 61± 11 48 ± 8 30 ± 6 Gemcitabine + expect. obs.* 3.0 91 ± 8 86 ± 1693 ± 16 70 ± 18 72 ± 11 50 ± 9 41 ± 10 30 ± 10 no 3 μM Se (X261, X295)expect.** 77 ± 17 66 ± 23 73 ± 14 76 ± 14 56 ± 12 44 ± 10 34 ± 8Gemcitabine + expect. obs. 30.0  80 ± 13 74 ± 12 57 ± 7 58 ± 7 40 ± 8***24 ± 7 27 ± 4 partial 30 μM Se (X261) expected 68 ± 17 58 ± 23 64 ± 1566 ± 15 49 ± 14 38 ± 12 expect. obs. 30.0 49 ± 6 55 ± 12 41 ± 10 50 ± 923 ± 7 23 ± 6 11 ± 6 10 ± 3 partial (X295) expected 43 ± 14 36 ± 21 39 ±13 41 ± 13 30 ± 12 24 ± 9 18 ± 7 *experimentally observed T/C values forthe combination of gemcitabine and selenium **expected T/C values forthe combination, calculated by the method: T/C_(expected) = T/C_(se) ×T/C_(drug)/100; T/C:T/C value in % with a given treatment at a specificconcentration ***synergistic drug concentrations are marked in italics.

TABLE 4c Inhibition of colony growth of the pancreatic cancer xenograftPAXF 736 by Na₂SeO₃ or mitomycin C alone or in combination in vitroSodium selenite Conc. T/C in T/C in % at a mitomycin C concentration(μM) of Treatment in μM % 1 × 10⁶ 1 × 10⁻⁵ 1 × 10⁻⁴ 1 × 10⁻³ 1 × 10⁻² 1× 10⁻¹ Synergism Mitomycin C (X261, X295) 77 ± 13 58 ± 10 56 ± 11 46 ± 830± 7 11 ± 7 Mitomycin C + expect. obs.* 3.0 81 ± 12 90 ± 15 60 ± 11 56± 8 48 ± 7 29 ± 3 10 ± 3 no 3 μM Se (X265, X290) expect.** 70 ± 16 53 ±13 51 ± 13 42 ± 12 27 ± 9 10 ± 9 Mitomycin C + expect. obs. 30.0 43 ± 8 25 ± 10 15 ± 7 27 ± 7 17 ± 6 19 ± 6  4 ± 3 no 30 μM Se (X265) expected33 ± 14 25 ± 11 24 ± 12 20 ± 9 14 ± 8  5 ± 5 expect. obs. 30.0 5 ± 2  4± 2  3 ± 2  3 ± 3  2 ± 2  1 ± 1  1 ± 1 n.d.*** (X290) expected*experimentally observed T/C values for the combination of mitomycin Cand selenium **expected T/C values for the combination, calculated bythe multiplication method: T/C_(expected) = T/C_(se) × T/C_(drug)/100;T/C:T/C value in % with a given treatment at a specific concentration***not determined

TABLE 4d Inhibition of colony growth of the pancreatic cancer xenograftPAXF 736 by Na₂SeO₃ or gemcitabine alone or in combination in vitroSodium selenite Conc. T/C in T/C in % at a gemcitabine concentration(μM) of Treatment in μM % 1 × 10⁶ 1 × 10⁻⁵ 1 × 10⁻⁴ 1 × 10⁻³ 1 × 10⁻² 1× 10⁻¹ Synergism Gemcitabine (X261, X295) 89 ± 11 78 ± 14 84 ± 12 77 ±13 71± 10 30 ± 8 Gemcitabine + expect. obs.* 3.0 81 ± 12 79 ± 13 82 ± 1284 ± 12 86 ± 15 66 ± 10 18 ± 8 no 3 μM Se (X265, X290) expect.** 72 ± 1263 ± 16 68 ± 13 62 ± 14 58 ± 12 24 ± 9 Gemcitabine + expect. obs. 30.043 ± 8  64 ± 15 62 ± 20 59 ± 13 39 ± 7 34 ± 6 14 ± 5 no 30 μM Se (X265)expected 38 ± 12 34 ± 15 36 ± 13 33 ± 14 31 ± 11 13 ± 9 expect. obs.30.0 5 ± 2  9 ± 7  5 ± 3  4 ± 3  5 ± 3  3 ± 2  1 ± 1 n.d.*** (X290)expected *experimentally observed T/C values for the combination ofgemcitabine and selenium **expected T/C values for the combination,calculated by the multiplication method: T/C_(expected) = T/C_(se) ×T/C_(drug)/100; T/C:T/C value in % with a given treatment at a specificconcentration ***not determined

TABLE 5a Synergistic effects of selenium in combination with gemcitabinein vitro Xeno- Se conc. Synergism with gemcitabine [μm] graft Exp. no.[μM] 1 × 10⁻⁶ 1 × 10⁻⁵ 1 × 10⁻⁴ 1 × 10⁻³ 1 × 10⁻² 0.1 1.0 PAXF 546 X251,X295 3.0 −* − − − − − − X261 30.0 − − − +* + + n.d. X295 30.0 − −− + + + + PAXF 736 X265, X290 3.0 − − − − − − n.d. X265 30.0 − − − − − −n.d. X290 30.0 not evaluable

TABLE 5b Synergistic effects of selenium in combination with mitomycin Cin vitro Xeno- Se conc. Synergism with mitomycin C [μm] graft Exp. no.[μM] 1 × 10⁻⁶ 1 × 10⁻⁵ 1 × 10⁻⁴ 1 × 10⁻³ 1 × 10⁻² 0.1 1.0 PAXF 546 X251,X295 3.0 −* − − − − − − X261 30.0 − − − − +* + n.d. X295 30.0 − − −− + + + PAXF 736 X265, X290 3.0 − − − − − − n.d. X265 30.0 − − − − − −n.d. X290 30.0 not evaluable *The experimentally determined T/C valuefor a drug combination is markedly lower (+) or not lower (−) than theexpected T/C value for this combination: n.d.: not determined.

In FIGS. 2 and 3 the effect of sodium selenite and mitomycin C isdemonstrated on the in vitro growth of PAXF 546. When 3 μM sodiumselenite were added to different concentrations of mitomycin C, nochange in the dose/response curve of mitomycin C could be observed, andtherefore no synergism between the two drugs occurs at this lowconcentration of selenite (FIGS. 2a and 3 a).

However, when 30 μM of sodium selenite were given simultaneously withmitomycin C, synergism was found at mitomycin C concentrations>0.01 μM(FIGS. 2b, c, 3 b, c and Table 5b).

Similar results were obtained with the combination of sodium seleniteand gemcitabine (FIGS. 4 and 5, Table 5a). Synergistic inhibition ofcolony formation in PAXF 546 was only found at a selenium concentrationof 30 μM and a gemcitabine concentration higher than 0.1 nM (FIG. 5,Table 5a).

In summary, the results show that high doses of selenium reduce thegrowth of many human tumor xenografts in vitro. Simultaneous treatmentof the pancreatic cancer cells PAXF 546 with 30 μM selenium andcytostatics, which directly react with the cellular DNA (e.g.gemcitabine and mitomycin C), results in a synergistic inhibition of thetumor growth in vitro.

3. Case Studies

The case documentations of five male tumor patients who received acombination therapy of highly dosed selenium (10-30 mg) and variouscytostatics are listed in the following. Two of the patients withpancreatic carcinoma (patient no. 4, 5) responded well to the therapydespite highly advanced diseases, also two patients (patient no. 1, 3)with hormone-resistant metastasized prostatic carcinoma. One patient(patient no. 2) with metastasized hypemephroma reacted with a partialremission to the therapy.

Patient No. 1

1.) Diagnosis: hormone-resistant metastasized prostatic carcinoma

2.) Diagnosed (month/year): June 1994

3.) Histology: cribriform prostatic carcinoma, degree III

4.) Tumor stage at the beginning of the therapy: T3, N2, M1

(T=extension of the primary tumor N=presence of lymph node metastasesM=presence of distant metastases)

5.) Localization of the distant metastases: lymph nodes, bones

6.) Tumor pretreatment: hormone therapy from VI/94 to XI/94

7.) Therapy with chemotherapy/selenium:

a) Chemotherapeutic agent: 5-fluorouracil, 750 mg, i.v., from XI/94 toV/95 mitomycin, 10 mg, i.v., from XI/94 to V/95

b) Selenium: Selenase®, 10 mg, i.v., from XI/94 to V/95

c) Response to the therapy: partial remission, complete regression oflymphatic edemata

d) Duration of the response: 7 months

e) Reason for terminating the therapy: progression

f) Therapy tolerance: good

8.) Preceding and concomitant diseases: coronary heart disease since1991, still existing at the beginning of the therapy

9.) Accompanying therapy during chemo/selenium therapy: Kerlone, Adalatfrom 1991 to V/95

10.) Survival status: dead

date of death: Aug. 31, 1994; due to tumor

Patient No. 2

1.) Diagnosis: metastasized hypemephroma

2.) Diagnosed (month/year): April 1996

3.) Histology: hypernephroma

4.) Tumor stage at the beginning of the therapy: T2, N1, M1

(T=extension of the primary tumor N=presence of lymph node metastasesM=presence of distant metastases)

5.) Localization of distant metastases: lung, liver

6.) Tumor pretreatment: nephrectomia 1996; interleukin II from IX/97 toI/98

7.) Therapy with chemotherapy/selenium:

a) Chemotherapeutic agent: Gemzar, 2 g, i.v., from I/98 to V/98

b) Selenium: Selenase®, 30 mg, i.v. from I/98 to V/98

c) Response to the therapy: partial remission

d) Duration of the response: so far 5 months

e) Reason for terminating the therapy: none

f) Therapy tolerance: good

8.) Preceding and concomitant diseases: polycythemia since 1990; stillexisting at the beginning of the therapy

9.) Accompanying therapy during chemo/selenium therapy: none

10.) Survival status: living; last observed May 18, 1998

Patient No. 3

1.) Diagnosis: metastasized hormone-resistant prostatic carcinoma

2.) Diagnosed (month/year): May 1997

3.) Histology: adenocarcinoma, degree II

4.) Tumor stage at the beginning of the therapy: T3, N1, M1

(T=extension of the primary tumor N=presence of lymph node metastasesM=presence of distant metastases)

5.) Localization of the distant metastases: bones

6.) Tumor pretreatment: orchiectomy at both sides 1997

7.) Therapy with chemotherapy/selenium:

a) Chemotherapeutic agent: adriblastine, 40 mg, i.v., from II/98 to V/98

b) Selenium: Selenase®, 30 mg, i.v., from II/98 to V/98

c) Response of the therapy: total remission

d) Duration of the response: so far 3 months

e) Reason for terminating the therapy: none

f) Therapy tolerance: good

8.) Preceding and concomitant diseases: none

9.) Accompanying therapy during chemo/selenium therapy: bisphosphonate(Bondronat) from II/98 to V/98

10.) Survival status: living, last observed May 20, 1998

Patient No. 4

1.) Diagnosis: pancreatic carcinoma

2.) Diagnosed (month/year): October 1994

3.) Histology: adenocarcinoma, degree II

4.) Tumor stage at the beginning of the therapy: T4, N1, M0

(T=extension of the primary tumor N=presence of lymph node metastasesM=presence of distant metastases)

5.) Localization of the distant metastases: none

6.) Tumor pretreatment: explorative laparotomy 1994, gastroenterostomy

7.) Therapy with chemotherapy/selenium:

a) Chemotherapeutic agent: 5-fluorouracil, 750 mg, i.v., from X/94 toVI/95, mitomycin, 10 mg, i.v., from X/94 to VI/95

b) Selenium: Selenase® 10 mg, i.v., from X/94 to VI/95

c) Response to the therapy: total remission, freedom from pain, weightgain

d) Duration of the response: 9 months

e) Reason for terminating the therapy: VI/95 cerebral metastasis

f) Therapy tolerance: good

8.) Preceding and concomitant diseases: none

9.) Accompanying therapy during chemo/selenium therapy: none

10.) Survival status: date of death Aug. 29, 1995; due to tumor

Patient No. 5

1.) Diagnosis: pancreatic carcinoma and gastric carcinoma

2.) Diagnosed (month/year): November 1997

3.) Histology: adenocarcinoma, degree III

4.) Tumor stage at the beginning of the therapy: T4, N1, M1

(T=extension of the primary tumor N=presence of lymph node metastasesM=presence of distant metastases)

5.) Localization of the distant metastases: liver, lymph nodes

6.) Tumor pretreatment:

a) Surgery: explorative laparotomy, gastroenterostomy 1997

b) Chemotherapy: Gemzar from XI/97 to I/98, high-dose 5-fluorouracil andleucovorins from I/98 to III/98, oxaliplatin from III/98 to IV/98

7.) Therapy with chemotherapy/selenium:

a) Chemotherapeutic agent: Gemzar, 1,2 mg, i.v., from IV/98 to V/98mitomycin, 10 mg, i.v. from IV/98 to V/98

b) Selenium: Selenase®, 30 mg, i.v., from IV/98 to V/98

c) Response to the therapy: partial remission, substantial painreduction

d) Duration of the response: 1 month

e) Reason for terminating the therapy: death

f) Therapy tolerance: pronounced stomatitis

8.) Preceding and concomitant diseases: none

9.) Accompanying therapy during chemolselenium therapy: none

10.) Survival status: dead

Date of death: May 13, 1998;

Cause of death: pancytopenia

What is claimed is:
 1. A method of producing a synergistic cytotoxiceffect on a cancer cell in a patient being treated for cancer,comprising contacting the cell with a cytostatic agent or a mixture ofcytostatic agents that inhibits mitosis and simultaneously, separatelyor sequentially contacting the cell with at least one salt of SeO₂, byadministering to the patient at least one salt of SeO₂ in aconcentration of 0.1 mg/kg body weight of the patient to 1.25 mg/kg bodyweight of the patient, and the cytostatic agent in a concentration of 2mg/m² body surface of the patient to 240 g/m² body surface of thepatient, or by administering to the patient at least one salt of SeO₂ ina concentration of 0.1 mg/kg body weight of the patient to 0.3 mg/kgbody weight of the patient, and the cytostatic agent in a concentrationof 20 mg/m² body surface of the patient to 1000 mg/m² body surface ofthe patient, whereupon the synergistic cytotoxic effect of thecytostatic agent or the mixture of cytostatic agents and at least onesalt of SeO₂ on the cell is produced wherein the cancer cell issensitive to the cytostatic agent and the salt of SeO2.
 2. The method ofclaim 1, wherein the cancer is large-cell bronchial carcinoma.
 3. Themethod of claim 1, wherein the cancer is small-cell bronchial carcinoma.4. The method of claim 1, wherein the cancer is adenocarcinoma.
 5. Themethod of claim 1, wherein the cancer is pancreatic carcinoma.
 6. Themethod of claim 1, wherein the cancer is prostatic carcinoma.
 7. Themethod of claim 1, wherein the cancer is hypemephroma.